Original Article Up-regulation of mir-10a and down-regulation of mir-148b serve as potential prognostic biomarkers for osteosarcoma

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1 Int J Clin Exp Pathol 2016;9(1): /ISSN: /IJCEP Original Article Up-regulation of mir-10a and down-regulation of mir-148b serve as potential prognostic biomarkers for osteosarcoma Yongbo An, Hongxing Zhao, Jun Zhang, Tan Lu. Jinling Jia, Bin Zhao Department of Orthopedics, The First Affiliated Hospital of Xinxiang Medical University, Weihui , China Received October 17, 2015; Accepted November 26, 2015; Epub January 1, 2016; Published January 15, 2016 Abstract: Background: MicroRNAs (mirnas) play key roles in cancer progression. The purpose of this study was to investigate mir-10a and mir-148b expression in osteosarcoma tissues and to further explored the prognostic value of the two mirnas. Methods: Quantitative real-time PCR method was used to evaluate the expression levels of mir- 10a and mir-148b in osteosarcoma (OS) tissues and adjacent non-tumor tissues. Furthermore, we investigated to clarify the relationship of mir-10a and mir-148 with clinicopathological features and survival in OS patients. Results: Our result suggested that mir-10a expression was up-regulated in OS tissues compared to adjacent nontumor tissues. The expression level of mir-148b was down-regulated in OS tissues compared to adjacent non-tumor tissues. High expressions level of mir-10a and low expressions level of mir-148b were significantly association with advanced TNM stage, and metastasis (P<0.05). Furthermore, Kaplan-Meier survival analysis and log-rank test indicated that high expressions of mir-10a and low expression of mir-148b were correlated with shorter overall survival of OS patients (log-rank test, P<0.05). Multivariate Cox proportional hazards model showed that increased expression of mir-10a, decreased expression of mir-148b, TNM stage, and metastasis were independent prognostic markers of overall survival of patients. Conclusion: Our data showed that high mir-10a level and low mir-148b level were correlated with aggressive progression and poor prognosis of OS. These mirnas may have clinical potentials as non-invasive diagnostic biomarkers for OS patients. Keywords: mir-10a, mir-148b, osteosarcoma, prognosis Introduction Osteosarcoma (OS) is one of the most malignant bone tumor in children and adolescents, which is characterized by formation of neoplastic bone tissue [1, 2]. Despite the advances in multiple therapeutic strategies, such as chemotherapy, surgery, and sometimes radiotherapy, overall clinical outcomes for osteosarcoma patients are still dissatisfactory [3, 4]. The 5-year cumulative survival rate for osteosarcoma patients was only 50-60% [5]. Therefore, the discovery of new biomarkers for the diagnosis, prognosis, and treatment of OS remains important. MicroRNAs (mirnas) are small non-coding RNAs nucleotides in length which regulate gene expression through repressing translation and cleaving their target mrnas by binding to complementary sites in their 3 -untranslated region (3 -UTR) [6]. Increasing evidence demonstrated that mirnas were involved in tumor genesis and cancer progression [7]. MiRNAs can function as either tumor suppressors or oncogenes according to their target genes in OS [8]. For example, Liu et al showed that mir-132 inhibited cell growth and metastasis in osteosarcoma cells by downregulation of Sox4 [9]. Jin et al found that mir-539 could suppress osteosarcoma cell invasion and migration by targeting MMP8 [10]. Zhu et al reported that mir-221 promote osteosarcoma cell proliferation, invasion and migration partly through the downregulation of PTEN [11]. Pan et al revealed that mir-27a promoted proliferation, migration and invasion by targeting MAP2K4 in human osteosarcoma cells [12]. These studies suggested that micrornas can play important roles in OS progression.

2 Table 1. The relationship of mir-10a and mir-148b expression with clinicopathological features of patients with osteosarcoma Clinicopathological features Number mir-10a expression mir-148b expression P value P value Low High Low High (mir-10a ) (mir-148b) Age (years) Children + Adolescents Young adults Gender Male Female Tumor size (cm) < Differentiation Well + moderate Poor TNM stage I + II III + IV Metastasis No Yes Figure 1. Expression levels of mir-10a and mir- 148b in osteosarcoma tissues and adjacent nontumor tissues.*p<0.05. In the present study, we examined the expression level of mir-10a and mir-148b in OS tissue samples using quantitative real-time PCR (qrt-pcr). Furthermore, we explored the association of mir-10a and mir-148b level with clinicopathological features and prognosis in OS patients. Materials and methods Patients and specimens A total of 41 samples were collected from patients with osteosarcoma and adjacent nontumor tissues between 2008 and 2011 from patients who were undergoing surgery in Department of Orthopedics, The First Affiliated Hospital of Xinxiang Medical University. None of the patients enrolled in this study had received chemotherapy or radiotherapy before surgery. All the samples were snap-frozen in liquid nitrogen and then stored at -80 C until use. Moreover, the diagnosis and the histological grading were confirmed by pathologists. The clinicopathological features are summarized in Table 1. The present study was approved by the Ethics Committee of the First Affiliated Hospital of Xinxiang Medical University; informed consent was obtained from all of the patients. Quantitative real-time PCR Total RNA was extracted using mirneasy kit (Qiagen) according to the manufacturer s instructions. The expression levels of mirnas quantitated using the TaqMan mirna assay kit (Applied Biosystems). Real-time PCR was performed using Rotor Gene 6000 Real-Time PCR (Qiagen) with an Invitrogen kit and a TaqMan universal PCR master mix. The relative expression levels of mirnas were normalized to that of internal control U6 by using 2 -ΔΔCt method. 187 Int J Clin Exp Pathol 2016;9(1):

3 Figure 2. Correlation between mirnas expression levels and survival in OS patients. Statistics analyses All computations were evaluated using SPSS 18.0 version for Windows. Data were expressed as means ± standard deviation (SD). Associations between mirnas expression level and clinicopathological features were determined using the Chi-square test. Moreover, logrank test and Kaplan-Meier method were used to evaluate survival rate in patients with osteosarcoma. Multivariate analysis was performed to evaluate prognostic values using Cox proportional hazards model. P<0.05 was considered statistically significant. Results We explored the expression level of mir-10a and mir- 148b expression levels in OS tissues using quantitative real-time PCR (qrt-pcr). We found that mir-10a expression was significantly increased in OS tissues than that in adjacent non-tumor tissues (P<0.05; Figure 1). On the other hand, mir-148b expression was significantly decreased in OS tissues than that in adjacent non-tumor tissues (P<0.05; Figure 1). For evaluation of the correlation between mir-10a/mir- 148b expression levels and the clinicopathological features, we categorized the patients into low expression group and high expression group based on the median expression level. Our data showed that high expression of mir-10a was significantly correlated with advanced TNM stage, and metastasis (P<0.05; Table 1). No significant difference was found between mir-10a and age, gender, tumor size, and differentiation (P>0.05; Table 1). On the other hand, decreased expression of mir- 148b was associated with advanced TNM stage, and metastasis (P<0.05; Table 1). Nevertheless, we did not find a significant correlation between mir-148b expression and other clinicopathological features (P>0.05; Table 1). In addition, Kaplan-Meier survival and log-rank test analysis showed that the high expressions of mir-10a and low expression of mir-148b were correlated with shorter overall survival (P<0.05; Figure 2A and 2B). Furthermore, Multivariate Cox proportional hazards model analysis revealed that increased expression of mir-10a and decreased expression of mir-148b, advanced TNM stage, and metastasis were independent prognostic fac- 188 Int J Clin Exp Pathol 2016;9(1):

4 Table 2. Multivariate analysis of the correlation of prognosis mir-10a with clinicopathological features Clinicopathological features HR 95% CI P value Age Gender Tumor size (cm) Differentiation TNM stage Metastasis mir-10a expression Table 3. Multivariate analysis of the correlation of prognosis mir-148b with clinicopathological features Clinicopathological features HR 95% CI P value Age Gender Tumor size (cm) Differentiation TNM stage Metastasis mir-148b expression tors for overall survival of OS patients (P<0.05; Tables 2 and 3). Discussion Dysregulation of mirnas was reported to be associated with the progression of human malignancies [13]. Aberrant expression of mir- NAs has been suggested to be as potential biomarkers for diagnosis and prognosis of osteosarcoma [14]. Thus, determination of functional and clinical importance of specific mirnas may provide effective management of OS. In the present study, the clinical importance of mir- 10a and mir-148b in OS were explored and the relationship with clinicopathological features was also determined. Our data showed that mir-10a was increased in OS tissues compared to the adjacent nontumor tissues. Moreover, the high expression level of mir-10a was associated with advanced TNM stage, and metastasis. Kaplan-Meier survival and log-rank test analysis showed that high expression of mir-10a was associated with shorter overall survival. Multivariate Cox proportional hazards model analysis indicated that up-regulated expression of mir-10a, advanced TNM stage, and metastasis were independent prognostic factors for overall survival in OS patients. Dysregulation of mir-10a expression has been also reported in kinds of tumors. For example, Safari et al showed that mir-20a expression was significantly up-regulated in cervical cancer and correlated with aggressive progression and poor prognosis of cervical cancer [15]. Yu et al suggested that mir- 10a was increased in NSCLC and associated with tumor node metastasis and lymph node metastasis. Furthermore, they found that mir-10a could promote NSCLC cell proliferation, migration and invasion by targeting PTEN [16]. Yan et al suggested that mir-10a was increased in glioma and promoted cell migration and invasion by negatively regulating the expression of EphA8 [17]. However, Khan et al reported that mir-10a expression was significantly decreased in breast cancer and regulated in part through retinoic acid [18]. Our study indicated that mir-10a could act as a tumor oncogene in OS progression. Furthermore, we found that mir-148b expression was decreased in OS tissues in comparison with adjacent non-tumor tissues and decreased expression of mir-148b was associated with high TNM stage, and metastasis. Furthermore, Kaplan-Meier survival and logrank test analysis demonstrated that decreased expression of mir-148b was correlated with shorter overall survival. The multivariate Cox proportional hazards model analysis indicated that down-regulated expression of mir-148b, high TNM stage, and metastasis were independent prognostic factors for overall survival in OS patients. Dysregulation expression of mir- 148b has been reported in types of tumors. For example, Ghasemkhani et al showed that down-regulated mir-148b expression as predictive biomarker and its prognostic significance associated with clinicopathological features in NSCLC patients [19]. Azizi et al revealed that mir-148b could act as a tumor suppressor genes through suppression of DNA methyltransferase-1 gene in pancreatic cancer cell lines [20]. Zhang et al found that mir-148b could suppress cell proliferation and invasion in hepatocellular carcinoma by targeting WNT1/ β-catenin pathway [21]. Our data expanded the tumor suppressive role of mir-148b in OS progression. 189 Int J Clin Exp Pathol 2016;9(1):

5 In conclusion, these results showed that the expression level of mir-10a and mir-148b contributed to aggressive progression of OS. MiR- 10a and mir-148b may serve as non-invasive prognostic biomarkers for OS patients. However, further studies are required to identify the role of these mirnas in progression and prognosis of osteosarcoma by their targets. Disclosure of conflict of interest None. Address correspondence to: Dr. Bin Zhao, Department of Orthopedics, First Affiliated Hospital of Xinxiang Medical University, Weihui, , China. References [1] Longhi A, Errani C, De Paolis M, Mercuri M and Bacci G. Primary bone osteosarcoma in the pediatric age: state of the art. Cancer Treat Rev 2006; 32: [2] Luetke A, Meyers PA, Lewis I and Juergens H. Osteosarcoma treatment -where do we stand? A state of the art review. Cancer Treat Rev 2014; 40: [3] Marina N, Gebhardt M, Teot L and Gorlick R. Biology and therapeutic advances for pediatric osteosarcoma. Oncologist 2004; 9: [4] Grimer R, Taminiau A and Cannon S. Surgical outcomes in osteosarcoma. J Bone Joint Surg Br 2002; 84: [5] Mirabello L, Troisi RJ and Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program. Cancer 2009; 115: [6] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009; 136: [7] Esquela-Kerscher A and Slack FJ. OncomirsmicroRNAs with a role in cancer. Nat Rev Cancer 2006; 6: [8] Zhang B, Pan X, Cobb GP and Anderson TA. micrornas as oncogenes and tumor suppressors. Dev Biol 2007; 302: [9] Liu Y, Li Y, Liu J, Wu Y and Zhu Q. MicroRNA-132 inhibits cell growth and metastasis in osteosarcoma cell lines possibly by targeting Sox4. Int J Oncol 2015; 47: [10] Jin H and Wang W. MicroRNA-539 suppresses osteosarcoma cell invasion and migration in vitro and targeting Matrix metallopeptidase-8. Int J Clin Exp Pathol 2015; 8: [11] Zhu J, Liu F, Wu Q and Liu X. mir-221 increases osteosarcoma cell proliferation, invasion and migration partly through the downregulation of PTEN. Int J Mol Med 2015; 36: [12] Pan W, Wang H, Jianwei R and Ye Z. MicroRNA- 27a promotes proliferation, migration and invasion by targeting MAP2K4 in human osteosarcoma cells. Cell Physiol Biochem 2014; 33: [13] Croce CM. Causes and consequences of microrna dysregulation in cancer. Nat Rev Genet 2009; 10: [14] Jones KB, Salah Z, Del Mare S, Galasso M, Gaudio E, Nuovo GJ, Lovat F, LeBlanc K, Palatini J, Randall RL, Volinia S, Stein GS, Croce CM, Lian JB and Aqeilan RI. mirna signatures associate with pathogenesis and progression of osteosarcoma. Cancer Res 2012; 72: [15] Safari A, Seifoleslami M, Yahaghi E, Sedaghati F and Khameneie MK. Upregulation of mir- 20a and mir-10a expression levels act as potential biomarkers of aggressive progression and poor prognosis in cervical cancer. Tumor Biology 2015; [Epub ahead of print]. [16] Yu T, Liu L, Li J, Yan M, Lin H, Liu Y, Chu D, Tu H, Gu A and Yao M. MiRNA-10a is upregulated in NSCLC and may promote cancer by targeting PTEN. Oncotarget 2015; 6: [17] Yan Y, Wang Q, Yan XL, Zhang Y, Li W, Tang F, Li X and Yang P. mir-10a controls glioma migration and invasion through regulating epithelialmesenchymal transition via EphA8. FEBS Lett 2015; 589: [18] Khan S, Wall D, Curran C, Newell J, Kerin MJ and Dwyer RM. MicroRNA-10a is reduced in breast cancer and regulated in part through retinoic acid. BMC Cancer 2015; 15: 345. [19] Ghasemkhani N, Shadvar S, Masoudi Y, Talaei AJ, Yahaghi E, Goudarzi PK and Shakiba E. Down-regulated MicroRNA 148b expression as predictive biomarker and its prognostic significance associated with clinicopathological features in non-small-cell lung cancer patients. Diagn Pathol 2015; 10: 164. [20] Azizi M, Teimoori-Toolabi L, Arzanani MK, Azadmanesh K, Fard-Esfahani P and Zeinali S. MicroRNA-148b and microrna-152 reactivate tumor suppressor genes through suppression of DNA methyltransferase-1 gene in pancreatic cancer cell lines. Cancer Biol Ther 2014; 15: [21] Zhang JG, Shi Y, Hong DF, Song M, Huang D, Wang CY and Zhao G. MiR-148b suppresses cell proliferation and invasion in hepatocellular carcinoma by targeting WNT1/β-catenin pathway. Sci Rep 2015; 5: Int J Clin Exp Pathol 2016;9(1):